It is to be noted that throughout this application, various publications are referenced by Arabic numerals in brackets. Full citation corresponding to each reference number is listed at the end of the specification. The disclosures of these publications are herein incorporated by reference in their entirety in order to describe fully and clearly the state of the art to which this invention pertains.
Acute renal failure (ARF) is a common ailment in patients admitted to the general medical-surgical hospitals. Furthermore, approximately half of the patients who develop ARF die, and survivors face marked increases in morbidity and prolonged hospitalization [1]. Early diagnosis is critical because renal failure is often asymptomatic, and it requires careful tracking of renal function markers in the blood. Dynamic monitoring of renal functions of patients at the bedside is highly desirable in order to minimize the risk of acute renal failure brought about by various clinical, physiological, and pathological conditions [2-6]. It is particularly important in the case of critically ill or injured patients because a large percentage of these patients face the risk of multiple organ failure (MOF) resulting in death [7, 8]. MOF is a sequential failure of lung, liver, and kidneys and is incited by one or more severe causes such as acute lung injury (ALI), adult respiratory distress syndrome (ARDS), hypermetabolism, hypotension, persistent inflammatory focus, or sepsis syndrome. The common histological features of hypotension and shock leading to MOF include tissue necrosis, vascular congestion, interstitial and cellular edema, hemorrhage, and microthrombi. These changes affect the lung, liver, kidneys, intestine, adrenal glands, brain, and pancreas in descending order of frequency [9]. The transition from early stages of trauma to clinical MOF is marked by the extent of liver and renal failure and a change in mortality risk from about 30% to about 50% [10].
Currently, the renal function is determined commonly by crude measurements such as urine output and plasma creatinine levels [11-13]. These values are frequently misleading because the values are affected by age, state of hydration, renal perfusion, muscle mass, dietary intake, and many other clinical and anthropometric variables. In addition, a single value obtained several hours after sampling is difficult to correlate with other important physiologic events such as blood pressure, cardiac output, state of hydration and other specific clinical events (e.g., hemorrhage, bacteremia, ventilator settings and others). An approximation of glomerular filtration rate (GFR) can be made via a 24 hour urine collection, but this process requires 24 hours to collect, several more hours to analyze, and a meticulous bedside collection technique. Unfortunately, detecting a patient's GFR by this time may be too late to treat the patient and have any hope of saving the kidney. New or repeat data are equally cumbersome to obtain. Occasionally, changes in serum creatinine must be further adjusted based on the values for urinary electrolytes, osmolality, and derived calculations such as the “renal failure index” or the “fractional excretion of sodium.” These require additional samples of serum collected contemporaneously with urine samples and, after a delay, precise calculations. Frequently, dosing of medication is adjusted for renal function and thus can be equally as inaccurate, equally delayed, and as difficult to reassess as the values upon which they are based. Finally, clinical decisions in the critically ill population are often equally as important in their timing as they are in their accuracy. Thus, there is a need to develop improved devices and methods for measuring GFR using non-ionizing radiation. The availability of a real-time, accurate, repeatable measure of renal excretion rate using exogenous markers under specific yet changing circumstances would represent a substantial improvement over any currently available or widely practiced method. Moreover, since such a method would depend solely on the renal elimination of the exogenous chemical entity, the measurement would be absolute and requires no subjective interpretation based on age, muscle mass, blood pressure, etc. In fact, if such a method were developed, it would represent the nature of renal function in the particular patient, under particular circumstances, at a precise moment in time.
Hydrophilic, anionic substances are generally recognized to be excreted by the kidneys [14]. Renal clearance occurs via two pathways, glomerular filtration and tubular secretion; the latter requires an active transport process, and hence, the substances clearing via this pathway are expected to possess very specific properties with respect to size, charge, and lipophilicity. It is widely accepted that the level of GFR represents the best overall measure of kidney function in the state of health or illness [15]. Fortunately, however, most of the substances that pass through the kidneys are filtered through the glomerulus. The structures of typical exogenous renal agents are shown in FIGS. 1 and 2. Substances clearing by glomerular filtration (hereinafter referred to as ‘GFR agents’) comprise inulin (1), creatinine (2), iothalamate (3) [16-18], 99mTc-DTPA (4), and 51Cr-EDTA (5), those undergoing clearance by tubular secretion include 99mTc-MAG3 (6) and o-iodohippuran (7) [16, 19, 20]. Among these, inulin is regarded as the “gold standard” for GFR measurement. All the compounds shown in FIGS. 1 and 2, except creatinine, require radioisotopes for detection.
As would be evident to one skilled in the art, cursory inspection of structures 1-7 provides no insight to ascertain the subtle factors responsible for directing the molecule to clear via a particular renal pathway. Clearly, gross physicochemical features such as charge, molecular weight, or lipophilicity are inadequate in even explaining the mode of clearance. Inulin (1, MW˜5000) and creatinine (2, MW 113) are both filtered through the glomerulus. On the other hand, the anionic chromium complex 5 (MW 362) and technetium complex 6 (MW 364) are cleared by different pathways. Structure-activity relationship (SAR) data on this very limited set of compounds is insufficient to ascertain the subtle differences between the two clearance pathways. Therefore, at the time of instant invention, prior art publications could not be relied upon to provide sufficient teaching or motivation for rational design of novel GFR agents. Thus, each new compound must be tested and compared against a known GFR agent, such as 99mTc-DTPA (4) or inulin (1), to confirm the clearance pathway.
As mentioned before, most of the currently known exogenous renal agents are radioactive. Currently, no reliable, continuous, repeatable bedside method for the assessment of specific renal function using non-radioactive exogenous GFR agent is commercially available. Among the non-radioactive methods, fluorescence measurement offers the greatest sensitivity. In principle, there are two general approaches for designing fluorescent GFR agents. The first approach involves enhancing the fluorescence of known renal agents (e.g. lanthanide or transition metal complexes) that are intrinsically poor emitters; and the second one involves transforming highly fluorescent conventional dyes, which are intrinsically lipophilic, into hydrophilic, anionic species to force them to clear via the kidneys. The present invention focuses on the former approach. Metal complexes of DTPA, DTPA-monoamides, DTPA-bisamides, and DTPA substituted at the ethylene portion of the ligand, have been used extensively in biomedical applications, and have been shown to clear through the kidneys. Work described in [21, 22, and 23] have independently suggested the use of luminescent metal complexes derived from polyaminocarboxylate ligands for measuring renal clearance.
The method of enhancing the fluorescence through intramolecular energy transfer process is well established [24], and has been applied to boost the fluorescence of metal ion through ligand-metal energy transfer [25-28]. The method essentially involves designing metal complexes containing an “antenna”. As used herein, an antenna is a moiety that has high photon capture cross section placed at an optimal distance (referred to as ‘Foster’ distance) from the metal ion wherein the moiety has a large surface area and a polarizable electron cloud. The distance between the antenna and the metal ion ranges from about 2-20 Å, preferably, from about 3-10 Å.
Novel fluorescent DTPA complexes for use in improved methods for providing data related to organ functioning are described below. These complexes may be said by some to be capable of real-time, accurate, repeatable measure of renal excretion rate.